Fig. 4: GII.4 interacts with ALIX and TSG101, with ALIX being critical for virus entry.

a GII.4 VLP, S and P domain binding to immobilized ALIX by ELISA. Error bars indicate mean ± SD with three replicates. b GII.4 VLP, S and P domain binding to immobilized TSG101 by ELISA. Error bars indicate mean ± SD with three replicates. c Bio-Layer Interferometry (BLI) to determine the binding affinity of GII.4 S and P domains to biotinylated ALIX and TSG101 (bALIX and bTSG101). d Effect of blocking virus interaction with ALIX and TSG101 on viral replication using specific pAbs at 1 h (gray) and 24 h (pink). Error bars indicate mean ± SD calculated using 2 HIE replicates for 1 h and 4 HIE replicates for 24 h (with two technical replicates/sample). e Confocal microscopy to probe GII.4-ALIX colocalization on the cell surface by detecting VP1 (green) and ALIX (red) using Gp Syd-pAb and rabbit anti-ALIX pAb. f ELISA to evaluate binding of GII.4 S- and mutant ∆S-domains to immobilized ALIX. Error bars indicate mean ± SD with 3 replicates. g Endocytosis induced by wild type GII.4 VLP and GII.4 VLP lacking the ALIX–binding motif (Δ VLP) using FM1-43FX in HIEs at 37 ^oC (n = 3 HIE replicates). h Viral replication in the presence of GII.4 WT VLPs (pink bars) and Δ VLPs (blue bars) was compared to untreated (black bar) at 24 h (blue dots). 1 h (gray dots) represents bound virus. Error bars indicate mean ± SD calculated using 2 HIE replicates for 1 h and 3 HIE replicates for 24 h (each condition with two technical replicates). i GII.4 replication in WT J2 and J2^ALIX-KD HIE monolayers indicated by percent fold change in viral RNA using the 2 − ΔΔCT method. Error bars indicate mean ± SD calculated using 3 HIE replicates for each condition with two technical replicates. All the experiments were repeated independently three times with similar results. P values for d, h, and i, relative to untreated control were calculated using one-way ANOVA, Dunnett’s multiple comparisons test. Source data are provided as a Source Data file.